Significant health risk exists due to increasing antibiotic-resistant bacteria and formation of antibiotic-resistant bacterial biofilm in water. For comprehensive understanding of stress response mechanisms of biofilm formation under sub-lethal photocatalysis (PC), Herein, a drip flow device was developed to culture biofilm and Pseudomonas aeruginosa (GEN) (resistance to Gentamicin) was developed to assess the effect of sub-lethal PC on biofilm formation. Under sub-lethal PC, the bacterial abundance and the thickness of biofilm decreased relative to controls (by 81.7% and 68.0%, respectively) on the 1st day; however, the content of extracellular polymeric substance (EPS) per cell was promoted unexpectedly. Furthermore, it was found that the proportion of viable culturable bacteria in the biofilm increased 7.8 times relative to control and the biofilm exhibited resistance to oxidative stress via EPS secretion. This perspective was validated through molecular regulatory network study. This study may provide enlightenment to bacterial biofilm control in the water system.